Evaluation of the specifity of hTERT gene induction by C0057684:

C0057684 upregulates multiple genes at Chromosome 5p15.33

Lancer K. Brown, Penelope Burke, Larissa Tersteege, Jessica Wheeler, William H. Andrews, Mieczyslaw A. Piatyszek, Laura A. Briggs, and Christopher A. Foster

Sierra Sciences, LLC, 250 S. Rock Blvd. Suite 130, Reno, NV 89502

www.sierrasci.com

Abstract

The inability to maintain telomere length in normal human cells lacking human telomerase (hTERT) activity results in replicative senescence. The identification of small molecules that induce telomerase activity in these normal cells may be useful for basic research aimed at studying telomerase regulation. Additionally, molecules found to induce hTERT expression, with high target selectivity, can possibly be used in therapeutic applications directed to alleviate or delay symptoms of aging. A high throughput screening (HTS) effort by Sierra Sciences LLC of a chemical library of 312,000 small molecules resulted in the identification of several compounds that can induce hTERT transcription in normal human fibroblasts. One of these, C0057684, was the subject of detailed evaluation in secondary assays. A critical objective was to clarify if C0057684 is a specific hTERT inducer, or if it has a broader effect on transcriptional activity at Chromosome 5p15.33. To examine expression levels of genes upstream and downstream of the hTERT locus after treatment with C0057684, we used Real-Time PCR on 20 different transcripts representing all the genes in the immediate proximity of the hTERT locus. Several genes, in addition to hTERT, were found to have increased transcription levels upon treatment with C0057684. Treatment of MRC5 cells with C0057684 did not, however, result in a decrease in transcription levels of any evaluated genes. Interestingly, there was no correlation between transcriptional activity and positions or orientations of genes relative to hTERT. Though our goal was to find compounds that specifically induce expression of hTERT, our data shows that C0057684 affects expression of other genes as well.

Summary

• A small study of gene expression at Chromosome 5p15.33 was carried out to clarify whether C0057684 is a specific hTERT inducer or a broad gene activator.
• Twenty genes upstream and downstream of the hTERT locus were tested.
• Five genes (PLEKHG4B, SLC9A3, TPPP, SLC6A18, SLC6A3) other than hTERT showed significant (greater than 16 fold) increases in transcription levels.
• None of the genes tested showed a significant decrease in transcription level.
• Position and orientation (relative to hTERT) appears to have little effect on the change in expression levels.
• We conclude that C0057684 has less than desirable specificity in the Chromosome 5p15.33 region.
• A larger DNA array study of changes in gene expression due to C0057684 across all chromosomes has been completed.

(see "Wang, T. et al. Gene Expression Profiling of MRC5 Lung Fibroblasts Treated with TA-65 and C0057684 using DNA Microarrays. UABBA conference, Los Angeles, 2008.")